The increasing human pressure exerted along coastlines and the subsequent increase in the delivery of pollutants at sea is a matter of concern worldwide. Urban wastewaters contain a variety of pollutants ... [more ▼]

The increasing human pressure exerted along coastlines and the subsequent increase in the delivery of pollutants at sea is a matter of concern worldwide. Urban wastewaters contain a variety of pollutants (mainly N, P, and trace elements) which can be involved in the launching of eutrophication. This complex process is able to fundamentally alter the integrity of coastal ecosystems thereby impairing the sustainability of economic activities and involving health risks for human through the consumption of sea products. Eutrophication is considered for more than 40 years as a pervasive process and a priority issue for the preservation of the health status of coastal ecosystems. The Mediterranean Sea supports high economic pressures in relation with the continuously increasing number of inhabitants and tourists in coastal areas. The physico-chemical (e.g. oligotrophy, microtidal regime) features of waters make them sensitive to eutrophication and several heavily urbanized areas have been experiencing adverse effects of this process for decades (e.g. biodiversity losses, Harmful Algal Blooms, fishes’ kills,…etc). Nowadays, smaller localities discharging insufficiently treated or raw wastewaters at sea also begin to report eutrophication problems especially during summer months when the number of tourists considerably increases the resident populations. National monitoring programs generally focus on priority areas by using either toxicological (e.g. trace metals in molluscs) and / or structural (e.g. phytoplankton biomasses) parameters to follow the evolution of already impacted water bodies. However, if the most deleterious effects of eutrophication are well known, little information is actually available regarding the early symptoms of the process. The identification of time- and cost-efficient indicators along with analytical procedures that would deliver early warning signals of pollution is therefore required to assist local authorities in the implementation of environmental policies. This research aimed at implementing some easy-to-use and efficient tools to detect the impact of urban wastewater pollution in Mediterranean coastal areas. A new set of potential early bioindicators has been identified. The gastropods Patella caerulea and Monodonta turbinata inhabiting the Mediterranean rocky midlittoral zone and epilithic biofilms were selected as good candidates for monitoring purpose. Biofilms which are microbenthic communities mainly composed of microalgae have been used for decades in freshwater systems as early indicators of pollution. In contrast, marine biofilms have largely been neglected and little is known regarding their composition, their physiology, and the way they react to wastewater pollution. We focused our studies on the Calvi Bay (NW Mediterranean, France) which is, regarding the anthropogenic pressure exerted on its coastal fringe, representative of other moderately urbanized areas of the Mediterranean basin. The Calvi Bay area is indeed among the preferred summer destinations by tourists in Corsica and is influenced by a single point source of pollution which is secondary-treated urban wastewater. A control vs. impacted sites approach has been used to assess the responses of the selected bioindicators to wastewater pollution. Our first task was to characterize the nature and amounts of the main pollutants (nutrients, trace elements) discharged in the Calvi Bay. Since nutrient measurements are commonly used to infer into the trophic status of water bodies, we assessed whether this parameter was reliable to detect the influence of wastewater discharge in the study area. The main pollutants discharged at sea were ammonium, phosphorus and iron. Amounts considerably increased during summer months (July and August) which was related to the high frequentation of the Calvi Bay area by tourists. Measurements of nutrient concentrations in seawater samples from controls and impacted sites did not allow evidencing the influence of wastewater inputs. In contrast, the high spatial and temporal resolution achieved through a small scale sampling design conducted in a small harbour impacted by wastewater discharges showed large spatial variability in the dispersion of effluents and hourly variations in the amounts of pollutants. The extrapolation of these results at the scale of the Calvi Bay may have explained our failure to detect the pollution by using samples collected punctually in space and time. These results suggested that the use of bioindicators that integrate the variable influence of nutrient pulses was required to evidence pollution. We focused our second study on a toxicological approach to detect the bioavailability of anthropogenically-derived nutrients in the midlittoral zone of the Calvi Bay and of the Marseille harbour. Multi-spatial scales and seasonal dual C and N stable isotope analyses were performed on the limpet Patella caerulea, the snail Monodonta turbinata, epilithic biofilms, and the macroalga Rissoella verruculosa. All bioindicators exhibited strongly elevated δ15N values at impacted sites compared to pristine ones, which revealed the influence of wastewater pollution in the midlittoral zone and the biological availability of anthropogenically-derived nitrogen at the base of the food web. Gastropods provided a time-integrated response reflecting the control vs. impacted status of sites. Results indicated that one sampling campaign per year should be sufficient to evidence wastewater pollution likely because of the slow turnover rate of gastropods’ muscles. Macroalgae showed a reliable but less consistent signal of wastewater pollution compared to other indicators. Only epilithic biofilms tended to show the occurrence of nutrient pulses during the tourist season which suggested that wastewater discharges may have influenced the composition and / or the physiology of communities. However the sampling of biofilms developed on natural rocky substrates was destructive and did not allow investigating the fine biological structure of communities and thus to fully understand the output of community scale parameters such as stable isotopes. We therefore recommended using biofilms grown on artificial substrates to circumvent this problem. We then allowed biofilms to develop on glass slides which are the most currently used artificial substrates in freshwater systems. Our task was to find out the most suitable technique to isolate and identify benthic diatoms which are common colonizers on newly available substrates. The species-specific tolerance to pollution of diatoms has been used for decades in the assessment of the health status of freshwater bodies. However, little is known on their marine counterparts and on their ability to evidence wastewater pollution. A specific and time-cost-efficient technique was implemented for the processing of lightly silicified benthic marine diatoms from Mediterranean oligotrophic areas. This was achieved through the multiple comparisons of existing protocols used either in sea- or fresh-waters. We finally investigated, by means of mesocosm deployed in situ and field experiments, the responses of biofilms developed on glass slides to a range of urban wastewater exposures. Colonization experiments lasted for 24 days in summer conditions. A multi-parametric assessment was conducted using a combination of toxicological and structural approaches applied to different biological scales. Toxicological parameters such as C-N stable isotopes, C:N:P ratios, and Trace Elements were measured at the community scale while the structural parameters were considered at the community (standing crops), assemblage (densities of the main autotroph and heterotroph organisms), and the genus (diatoms) scales. The mesocosm experiments were highly efficient to demonstrate the good potential of biofilms as early indicators of wastewater pollution. The impact of wastewater pollution was mainly identified at the community and the genus (diatoms) scales. Standing crops and the C-N stable isotopes were the most useful parameters showing respectively a stimulation of microalgal biomasses (i.e. eutrophication) and the bioavailability of wastewater-derived nitrogen even at low pollution levels. At the genus scale, the composition of diatoms’ assemblages changed markedly especially in the most polluted mesocosms. Results notably highlighted the proliferation of the small-sized individuals of Entomoneis which was thought to outcompete the larger diatoms belonging to the genus Mastogloia for nutrient uptake. The responses of biofilms to pollution largely differed between mesocosm and field experiments. Nevertheless, standing crop parameters corroborated results obtained in mesocosms allowing to assume an influence of wastewater pollution in the Calvi Bay. Field samples were generally characterized by the presence of well developed hydrozoan colonies which were only seldom reported at mesocosm sites. The presumably impacted site also exhibited the highest densities in other heterotrophic eukaryote groups (e.g. nematodes, polychaetes, foraminifers) and primary producers. We interpreted these results either as a top-down effect or as an increase in habitat complexity. The genus scale determination of diatoms’ assemblages showed a decrease in the relative abundances of Mastogloia at the impacted site which was in accordance with results from the mesocosm experiments. The highest density values were also observed for Cylindrotheca at impacted site. The last part of this research gathers the multiple spatial and temporal responses provided by the selected bioindicators to urban wastewater pollution in order to validate their future routine use in the context of monitoring programs. The occurrence of potential confusion sources in the interpretation of data was critically reviewed. The time-cost-efficiency of the tested parameters was then evaluated in order to assist environmental managers in their choices of biofilm-based techniques for detecting wastewater pollution. Finally since biofilms exhibited some strong accumulations of toxic elements, ecological hypotheses dealing with the trophic role of biofilms and the transfer of pollutants through the food webs are provided. [less ▲]

The processing of benthic diatoms is tedious and involves several potentially damaging steps for cells. Although the preservation of siliceous frustules is of paramount importance in the implementation of ... [more ▼]

The processing of benthic diatoms is tedious and involves several potentially damaging steps for cells. Although the preservation of siliceous frustules is of paramount importance in the implementation of biotic indices, only few studies quantified treatment-induced cell losses. We assumed that commonly used treatments may lead to mechanical (centrifugation, sedimentation, boiling, sonication and mounting in Naphrax) and chemical (cold H2O2 digestion) damages on diatoms. We analysed the potential adverse effects of these treatments and the cleaning efficiency of H2O2 and incineration in order to find out the most suitable technique to process lightly silicified Mediterranean populations. Results showed that successive resuspensions of material after each concentration treatment (sedimentation and centrifugation) and low speed centrifugation did not alter the physical integrity of frustules. In contrast, boiling and sonication exhibited adverse effects especially on the preservation of large frustules and Naphrax mounting proved to be the most damaging step whatever the size of diatoms. For cleaning treatments, incineration provided the most satisfactory results and acted on a non-selective way as opposed to hydrogen peroxide which led to either a large number of non-cleaned frustules or dissolved valves. Our recommendations for processing samples of lightly silicified Mediterranean benthic diatoms include the use of low speed centrifugations, dehydration at room temperature, incineration and dry mounting. [less ▲]

Increasing human pressure along Mediterranean coastlines raises the need to define sensitive bioindicators that provide an early response to nutrient enrichment. We performed multiscale carbon and ... [more ▼]

Increasing human pressure along Mediterranean coastlines raises the need to define sensitive bioindicators that provide an early response to nutrient enrichment. We performed multiscale carbon and nitrogen stable isotope analyses on the limpet Patella caerulea, the snail Monodonta turbinata, epilithic biofilms, and the macroalga Rissoella verruculosa inhabiting the rocky midlittoral zone. Samples were seasonally collected in 2006 from 5 sites exposed to a range of anthropogenic discharges in the Revellata Bay area and in Marseille harbour (France). All bioindicators exhibited strongly elevated δ15N values at impacted sites compared to pristine ones, which revealed the biological availability of anthropogenically derived nutrients. Only epilithic biofilms tended to show both the occurrence of nutrient pulses during the tourist season and a δ13C response at impacted sites. In contrast to macroalgae, which exhibited a somewhat equivocal signal, gastropods and especially M. turbinata provided the best time-integrated picture of the graduated exposure of the 5 sites to anthropogenic impact. Results also showed first evidence of large isotopic variability at a scale of tens of metres, close to that found at the kilometre scale. The intra- and interspecific isotopic variability in gastropods may be explained by the patchiness of resources and specific morphological and behavioural features, but these factors do not greatly hamper their potential as early bioindicators of wastewater disturbances. [less ▲]

Eutrophication is considered to be one of the main threats to marine environments leading to changes in trophic status of ecosystems and alteration of biological diversity. Carbon and Nitrogen stable ... [more ▼]

Eutrophication is considered to be one of the main threats to marine environments leading to changes in trophic status of ecosystems and alteration of biological diversity. Carbon and Nitrogen stable isotopes analyses were performed on the limpet Patella caerulea and one of its food sources (epilithic biofilms) to assess their potential as early indicators of eutrophication. Samples were seasonally collected in 2005-2006 on five locations gradually exposed to urban sewage in the Calvi Bay and in the Marseilles harbour. Stable isotope signatures of Patella caerulea muscles exhibited steady site - specific values over seasons. In contrast to this time – integrated signal, wide variations in biofilm values show that either composition or isotopic ratios of food sources may vary greatly in time and space. Elevated δ15N values of limpets and biofilms, typical of wastewater influence but unrelated to nitrogen loads, indicate the biological availability of sewage-derived nitrogen in the Calvi Bay and the Marseilles harbour. A reference level of δ15N values is rapidly reached with increasing depth that indicates the limited vertical extent of pollution in the Calvi Bay. [less ▲]